Identification of new compounds with high activity against stationary phase Borrelia burgdorferi from the NCI compound collection

Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi. Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi. This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti-Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives—sylvestrosides III and IV, that showed a considerable anti-Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.

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Evaluation of Disulfiram Drug Combinations and Identification of Other More Effective Combinations against Stationary Phase Borrelia burgdorferi

Antibiotics ◽

10.3390/antibiotics9090542 ◽

2020 ◽

Vol 9 (9) ◽

pp. 542 ◽

Cited By ~ 1

Author(s):

Hector Alvarez-Manzo ◽

Yumin Zhang ◽

Wanliang Shi ◽

Ying Zhang

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Stationary Phase ◽

Drug Exposure ◽

Drug Combinations ◽

Persistent Symptoms ◽

Exposure Experiment ◽

Vector Borne ◽

Treatment Alternatives ◽

Possible Cause

Lyme disease, caused by Borrelia burgdorferi, is the most common vector-borne disease in USA, and 10–20% of patients will develop persistent symptoms despite treatment (“post-treatment Lyme disease syndrome”). B. burgdorferi persisters, which are not killed by the current antibiotics for Lyme disease, are considered one possible cause. Disulfiram has shown to be active against B. burgdorferi, but its activity against persistent forms is not well characterized. We assessed disulfiram as single drug and in combinations against stationary-phase B. burgdorferi culture enriched with persisters. Disulfiram was not very effective in the drug exposure experiment (survival rate (SR) 46.3%) or in combinations. Clarithromycin (SR 41.1%) and nitroxoline (SR 37.5%) were equally effective when compared to the current Lyme antibiotic cefuroxime (SR 36.8%) and more active than disulfiram. Cefuroxime + clarithromycin (SR 25.9%) and cefuroxime + nitroxoline (SR 27.5%) were significantly more active than cefuroxime + disulfiram (SR 41.7%). When replacing disulfiram with clarithromycin or nitroxoline in three-drug combinations, bacterial viability decreased significantly and subculture studies showed that combinations with these two drugs (cefuroxime + clarithromycin/nitroxoline + furazolidone/nitazoxanide) inhibited the regrowth, while disulfiram combinations did not (cefuroxime + disulfiram + furazolidone/nitazoxanide). Thus, clarithromycin and nitroxoline should be further assessed to determine their role as potential treatment alternatives in the future.

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Borrelia burgdorferi Proteins Whose Expression Is Similarly Affected by Culture Temperature and pH

Infection and Immunity ◽

10.1128/iai.69.4.2739-2742.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2739-2742 ◽

Cited By ~ 53

Author(s):

Ramesh Ramamoorthy ◽

Dorothy Scholl-Meeker

Keyword(s):

Borrelia Burgdorferi ◽

Stationary Phase ◽

Growth Phase ◽

Environmental Conditions ◽

Stationary Growth Phase ◽

Culture Temperature ◽

Stationary Growth ◽

Encoding Genes ◽

Midgut Ph ◽

Tick Midgut

ABSTRACT Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, ofBorrelia burgdorferi in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed that the expression of each of the remaining stationary-phase-upregulated proteins, P35 included, was also influenced by culture temperature; these proteins were selectively expressed at 34°C but not at 24°C. Significantly, the expression of a majority of these proteins was also affected by culture pH, since they were abundantly expressed at pH 7.0 (resembling the tick midgut pH of 6.8 during feeding) but only sparsely at pH 8.0 (a condition closer to that of the unfed tick midgut pH of 7.4). We propose that this group of B. burgdorferi proteins, which in culture is selectively expressed under conditions of 34°C and pH 7.0, may be induced in the tick midgut during the feeding event. Furthermore, the differential and coordinate expression of these proteins under different environmental conditions suggests that the encoding genes may be coregulated.

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Essential Oils with High Activity against Stationary Phase Bartonella henselae

Antibiotics ◽

10.3390/antibiotics8040246 ◽

2019 ◽

Vol 8 (4) ◽

pp. 246 ◽

Cited By ~ 5

Author(s):

Xiao Ma ◽

Wanliang Shi ◽

Ying Zhang

Keyword(s):

Stationary Phase ◽

Essential Oils ◽

High Activity ◽

High Throughput Screening ◽

Bartonella Henselae ◽

Current Treatment ◽

Bacterial Cells ◽

Active Components ◽

Chronic Infections ◽

Cinnamon Bark

Bartonella henselae is a fastidious Gram-negative intracellular bacterium that can cause cat scratch disease, endocarditis in humans and animals, as well as other complications, leading to acute or chronic infections. The current treatment for Bartonella infections is not very effective presumably due to bacterial persistence. To develop better therapies for persistent and chronic Bartonella infections, in this study, with the help of SYBR Green I/PI viability assay, we performed a high-throughput screening of an essential oil library against the stationary phase B. henselae. We successfully identified 32 essential oils that had high activity, including four essential oils extracted from Citrus plants, three from Origanum, three from Cinnamomum, two from Pelargonium, and two from Melaleuca, as well as frankincense, ylang-ylang, fir needle, mountain savory (winter), citronella, spearmint, elemi, vetiver, clove bud, allspice, and cedarwood essential oils. The minimal inhibitory concentration (MIC) determination of these 32 top hits indicated they were not only active against stationary phase non-growing B. henselae but also had good activity against log-phase growing B. henselae. The time-kill assay showed 13 active hits, including essential oils of oregano, cinnamon bark, mountain savory (winter), cinnamon leaf, geranium, clove bud, allspice, geranium bourbon, ylang-ylang, citronella, elemi, and vetiver, could eradicate all stationary phase B. henselae cells within seven days at the concentration of 0.032% (v/v). Two active ingredients, carvacrol and cinnamaldehyde, of oregano and cinnamon bark essential oils, respectively, were shown to be very active against the stationary phase B. henselae such that they were able to eradicate all the bacterial cells even at the concentration ≤ 0.01% (v/v). More studies are needed to identify the active components of some potent essential oils, decode their antimicrobial mechanisms, and evaluate their activity against Bartonella infections in animal models.

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Extracellular Secretion of the Borrelia burgdorferi Oms28 Porin and Bgp, a Glycosaminoglycan Binding Protein

Infection and Immunity ◽

10.1128/iai.72.11.6279-6286.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6279-6286 ◽

Cited By ~ 22

Author(s):

Robert G. Cluss ◽

Damon A. Silverman ◽

Thomas R. Stafford

Keyword(s):

Borrelia Burgdorferi ◽

Stationary Phase ◽

Outer Membrane ◽

Regulatory Networks ◽

Binding Protein ◽

Mass Spectrometry Analysis ◽

Logarithmic Phase ◽

Spectrometry Analysis ◽

Rpmi Medium ◽

Extracellular Secretion

ABSTRACT Borrelia burgdorferi, the Lyme disease pathogen, cycles between its Ixodes tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of Borrelia burgdorferi strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 107 to 109 cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of B. burgdorferi protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered.

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Essential Oils with High Activity Against Stationary Phase Bartonella Henselae

10.20944/preprints201910.0178.v1 ◽

2019 ◽

Author(s):

Xiao Ma ◽

Wanliang Shi ◽

Ying Zhang

Keyword(s):

Stationary Phase ◽

Essential Oils ◽

High Activity ◽

High Throughput Screening ◽

Drug Exposure ◽

Bartonella Henselae ◽

Current Treatment ◽

Bacterial Cells ◽

Chronic Infections ◽

Cinnamon Bark

Bartonella henselae is a fastidious Gram-negative intracellular bacterium which can cause cat scratch disease, endocarditis in humans and animals as well as other complications, leading to acute or chronic infections. The current treatment for Bartonella infections is not very effective due to antibiotic resistance and also persistence. To develop better therapies for persistent and chronic Bartonella infections, in this study, with the help of SYBR Green I/PI viability assay, we performed a high-throughput screening of an essential oil library against stationary phase B. henselae. We successfully identified 32 essential oils that had high activity, including four essential oils extracted from Citrus plants, three from Origanum, three from Cinnamomum, two from Pelargonium and two from Melaleuca, as well as frankincense, ylang ylang, fir needle, mountain savory (winter), citronella, spearmint, elemi, vetiver, clove bud, allspice and cedarwood essential oils. The minimal inhibitory concentration (MIC) determination of these 32 top hits indicated they were not only active against stationary phase non-growing B. henselae but also had good activity against log phase growing B. henselae. The time-kill curve by drug exposure assay showed 13 active hits, including essential oils of oregano, cinnamon bark, mountain savory (winter), cinnamon leaf, geranium, clove bud, allspice, geranium bourbon, ylang ylang, citronella, elemi and vetiver, could eradicate all stationary phase B. henselae cells within 7 days at the concentration of 0.032% (v/v). Two active ingredients, carvacrol and cinnamaldehyde, of oregano and cinnamon bark essential oils, respectively, were shown to be very active against stationary phase B. henselae such that they were able to eradicate all the bacterial cells even at the concentration ≤ 0.01% (v/v). Our finding of active essential oils may help to develop more effective treatments for persistent Bartonella infections.

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1,2,4-Oxadiazole-Based Bio-Isosteres of Benzamides: Synthesis, Biological Activity and Toxicity to Zebrafish Embryo

International Journal of Molecular Sciences ◽

10.3390/ijms22052367 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2367

Author(s):

Sen Yang ◽

Chao-Li Ren ◽

Tian-Yang Ma ◽

Wen-Qian Zou ◽

Li Dai ◽

...

Keyword(s):

Biological Activity ◽

Antifungal Activity ◽

Acute Toxicity ◽

High Activity ◽

Broad Spectrum ◽

Zebrafish Embryo ◽

Thanatephorus Cucumeris ◽

Low Toxicity ◽

New Compounds ◽

Better Than

To discover new compounds with broad spectrum and high activity, we designed a series of novel benzamides containing 1,2,4-oxadiazole moiety by bioisosterism, and 28 benzamides derivatives with antifungal activity were synthesized. These compounds were evaluated against four fungi: Botrytis cinereal, FusaHum graminearum, Marssonina mali, and Thanatephorus cucumeris. The results indicated that most of the compounds displayed good fungicidal activities, especially against Botrytis cinereal. For example, 10a (84.4%), 10d (83.6%), 10e (83.3%), 10f (83.1%), 10i (83.3%), and 10l (83.6%) were better than pyraclostrobin (81.4%) at 100 mg/L. In addition, the acute toxicity of 10f to zebrafish embryo was 20.58 mg/L, which was classified as a low-toxicity compound.

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Differential Expression of Borrelia burgdorferi Proteins during Growth In Vitro

Infection and Immunity ◽

10.1128/iai.66.11.5119-5124.1998 ◽

1998 ◽

Vol 66 (11) ◽

pp. 5119-5124 ◽

Cited By ~ 65

Author(s):

Ramesh Ramamoorthy ◽

Mario T. Philipp

Keyword(s):

Borrelia Burgdorferi ◽

Stationary Phase ◽

Constitutive Expression ◽

Logarithmic Phase ◽

Dependent Manner ◽

Growth Phases ◽

Stationary Growth ◽

Early Stationary Phase ◽

Regulated Expression

ABSTRACT In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165–1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB,groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospCand bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.

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The Chemistry of Olive Oil: an endless story

OCL ◽

10.1051/ocl/2020024 ◽

2020 ◽

Vol 27 ◽

pp. 28

Author(s):

Lanfranco Conte

Keyword(s):

Stationary Phase ◽

Olive Oil ◽

Analytical Methods ◽

Degradation Products ◽

Analytical Control ◽

Chromatographic Separations ◽

Olive Oils ◽

Minor Compounds ◽

Short Time ◽

New Compounds

Because of their value, Olive oils, undergo to several attempts of adulteration and this improved the performances of analytical methods developed to check their authenticity. After the eve of the “Chemistry of indexes”, the development of separation techniques greatly improved the knowledge of the composition of lipids; the earlier gas chromatographic separations of fatty acid, could last even more than 40 minutes and the evaluation of minor fatty acids were problematic, at Bologna University, their previous separation on silver nitrate silica gel TLC was applied in 1974–1975, in order to concentrate them and obtain a better chromatogram. The gas chromatographic evaluation of minor compounds was another interesting challenge, packed columns with apolar stationary phase, admit the separation of few peaks; in 1975, the use of a slightly more polar stationary phase (OV17) highlighted the presence of Δ-5-avenasterol in olive oils. 1981 is a milestone for olive oil analytical control: Regulation (CEE) 2568/91 made the use of capillary columns mandatory. Official analytical methods consider many parameters, enclosed sensory evaluation, that is not matter for chemists, however, chemists are moved by curiosity and several papers had been published on the relationship between the volatile compounds of head space of olive oil and sensory characteristics. Despite being one of the more studied food, olive oils’ composition continues to give the chemists the possibility to discover new compounds, e.g. degradation products of sterols that leads to the standardization of the method for sterenes evaluation. From 1973 to nowadays, the laboratory performances greatly improved, nowadays it is possible to generate a huge number of data in a short time, then the problem is how to correctly interpretate them, surely chemometric and lipidomic greatly can help.

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